首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
The mechanism of the biosynthesis of albumin was studied in cell suspensions from rat liver. The cells were prepared by continuous perfusion of the liver in situ with 0.05% collagenase and 0.10% hyaluronidase and incubated under conditions optimized for the incorporation of amino acids into protein. Seven minutes after starting the incubation L-[1-14C]leucine was added, followed after 25 min by a 15 or 30-min chase with an 830-fold excess of non-radioactive L-leucine. Total protein, an albumin-like protein, and albumin were isolated from samples withdrawn immediately of total protein was found to remain constant after addition of the non-radioactive L-leucine, whereas that of the albumin-like protein decreased and that of albumin increased with incubation time. The increase in albumin radioactivity accounted for the decrease in radioactivity of the albumin-like protein, suggesting that the latter is a precursor of albumin. The precursor protein differed from albumin by an oligopeptide extension at the N-terminal end.  相似文献   

2.
1. A protein(s) of rat liver (precipitated from soluble extracts of the microsomal fraction by anti-albumin) yields albumin after limited hydrolysis by trypsin. 2. Evidence that the product of limited tryptic hydrolysis is albumin, is based upon ion-exchange chromatography, electrofocusing and peptide `mapping'. 3. The albumin `precursor' is recognized by anti-albumin and is apparently not distinguished from albumin by anti-albumin. 4. A small peptide is liberated from the presumptive albumin precursor during limited tryptic hydrolysis. This peptide is labelled by arginine, but not by leucine, lysine or methionine. 5. These results support our previous suggestion based on kinetic evidence that the albumin-like protein(s), in the anti-albumin precipitate from rat liver, is an albumin precursor.  相似文献   

3.
The separation of intracellular serum albumin from rat liver   总被引:6,自引:4,他引:2  
1. Antibody precipitation of serum albumin from rat liver extracts yields impure preparations of the protein. 2. When rat liver is labelled with l-[1-(14)C]leucine, antibody precipitation of albumin leads to material that is contaminated with a protein or proteins of very high specific radioactivity. Only 10-25% of the radioactivity of the antibody precipitate is associated with serum albumin. 3. A chromatographic procedure is described that can be used to separate radiochemically pure serum albumin from antibody precipitates obtained from extracts of rat liver. 4. Extracellular albumin secreted by liver slices yields a precipitate with antibody which contains much less radioactive impurity. About 70-90% of the radioactivity is associated with serum albumin. Serum albumin separated by antibody precipitation from rat serum labelled in vivo was not contaminated with the radiochemical impurities associated with intracellular albumin. 5. A simple method is described of obtaining the content of serum albumin in rat liver extracts by the technique of isotope dilution and ion-exchange chromatography.  相似文献   

4.
Immunologically isolated albumin from rat liver microsomes separates on DEAE-cellulose into almost equal proportions of an albumin-like protein and authentic albumin. Besides this similarity in immunological properties, both albumin species have almost the same molecular weight and amino acid composition. Furthermore, the amino acid sequences appear to be identical apart from an additional pentapeptide at the N-terminus of the albumin-like protein. It is suggested that the albumin-like protein represents a precursor which is converted to albumin by release of a pentapeptide from the N-terminus.  相似文献   

5.
Two hours after systemic injection of bovine plasma albumin (BPA) into pregnant mice, albumin-like antigen was detected by indirect immunohistological methods within the cytoplasm of oviductal and preimplantation uterine embryos whether ovulation was spontaneous or induced by hormone injection. Although fluorescence, localising antigen similar to or identical with the systemically injected foreign protein, was present in embryos in all oviductal regions and at all cleavage stages, the intraembryonic location of the transferred serum molecules differed with embryo stage. Most ootids and two-celled blastomeres contained large intracytoplasmic areas of intense fluorescence randomly associated with non-fluorescent or dimly fluorescent areas in the same cell. By four- and eight-celled stages, albumin-like antigen was localised at the periphery of blastomeres; less was found deep within embryos. By morula and blastocyst stages, blastomeres differed from each other in fluorescence intensity although intracellular fluorescence was homogeneous. Transferred BPA antigen, present in both pronuclei, probably was absent from blastomere nuclei. Ootid zonae pellucidae contained BPA antigen but none was detected in zonae surrounding cleaving embryos. Little foreign albumin was detected in oviductal epithelium.
It is concluded that morphological, as well as biochemical, differentiation occurs during mammalian cleavage and it is suggested that maternal macromolecular contributions to mammalian preimplantation embryos may be necessary for normal development in vivo .  相似文献   

6.
Two hours after systemic injection of bovine plasma albumin (BPA) into pregnant mice, albumin-like antigen was detected by indirect immunohistological methods within the cytoplasm of oviductal and preimplantation uterine embryos whether ovulation was spontaneous or induced by hormone injection. Although fluorescence, localising antigen similar to or identical with the systemically injected foreign protein, was present in embryos in all oviductal regions and at all cleavage stages, the intraembryonic location of the transferred serum molecules differed from embryo stage. Most ootids and two-celled blastomeres contained large intracytoplasmic areas of intense fluorescence randomly associated with non-fluorescent or dimly fluorescent areas in the same cell. By four- and eight-celled stages, albumin-like antigen was localised at the periphery of blastomeres; less was found deep within embryos. By morula and blastocyst stages, blastomeres differed from each other in fluorescence intensity although intracellular fluorescence was homogeneous. Transferred BPA antigen, present in both pronuclei, probably was absent from blastomere nuclei. Ootid zonae pellucidae contained BPA antigen but none was detected in zonae surrounding cleaving embryos. Little foreign albumin was detected in oviductal epithelium. It is concluded that morphological, as well as biochemical, differentiation occurs during mammalian cleavage and it is suggested that maternal macromolecular contributions to mammalian preimplantation embryos may be necessary for normal development in vivo.  相似文献   

7.
Biosynthesis of Rat serum albumin   总被引:5,自引:5,他引:0  
1. The labelling of intracellular and extracellular serum albumin was studied in liver slices and in whole rats by using new methods for the purification of the protein. 2. The results suggest that a polypeptide precursor is formed that is converted relatively slowly into serum albumin. 3. The effect of liver cell K(+) has been examined by a double-label method and it is shown that K(+) accelerates the rate of conversion of ;precursor' into albumin. The rate of transit of albumin across the cell membrane appears to be unrelated to the concentration of K(+) within the cell. 4. The time-course of incorporation of radioactive amino acid into albumin follows a sigmoidal mode. There is a pronounced time-lag before label starts to appear in intracellular albumin, and a further time-lag before it appears in extracellular albumin. 5. In slices the sum of intra- and extra-cellular label rises steadily from 30min after the start of labelling with a pulse of labelled leucine or valine and continues to rise for at least another 60min. This occurs whether labelling is stopped by addition of excess of carrier amino acid or with cycloheximide (100mum) or both. 6. The intracellular albumin content remains constant whether slices are maintained with low or normal intracellular K(+) concentrations. 7. Specific radioactivities of intracellular albumin (and fractions thereof) and of extracellular albumin were determined in vitro and in vivo. The results show that the intracellular albumin cannot be a precursor of extracellular albumin, unless a very small compartment is turning over much more rapidly than the bulk of the liver albumin or even of the microsomal albumin.  相似文献   

8.
1. The fractionation of intracellular albumin labelled with radioactive l-leucine was studied in rat liver by means of isoelectric focusing. 2. Isoelectric fractionation was compared with ion-exchange chromatography for purification of radioactive intracellular albumin obtained by antibody precipitation. Similar results were obtained with both methods of separation. Purified albumin contains only a minor amount of the radioactivity. The remainder is associated with albumin-like protein(s). 3. The albumin-like protein has the properties of a precursor of plasma albumin. 4. The distribution and turnover of radioactive albumin in rough and smooth microsomal fractions and in a Golgi-rich fraction were studied. 5. It is concluded that newly synthesized albumin, as such, appears only momentarily if at all in any intracellular structure before its appearance in the plasma. 6. It is also concluded that the rate-limiting step in the secretion of plasma albumin is the conversion of precursor(s) into albumin. We can find no evidence to suggest that there is any significant transport of albumin, as such, during the course of secretion.  相似文献   

9.
Abstract— The incorporation in vivo of [3H]leucine into protein from subcellular fractions was determined in rats chronically ingesting 15 per cent ethanol for 8 months. Mitochondrial, microsomal and cell sap fractions from cerebellum, cortex cerebri and liver were investigated. The results showed a minor over-all depression of protein synthesis in cerebellum and cortex cerebri and a slight stimulation of the incorporation of leucine into protein from liver subcellular fractions. If the animals were abstinent 24 h before injection of the isotope, the incorporation of labelled amino acids into protein was markedly increased in cerebellum and cerebral cortex but not in liver.  相似文献   

10.
1. The intravenous injection of puromycin to mice 0.5 min after administration of radioactive leucine resulted in the release of labelled ribosome-bound nascent protein chains with the next 0.5 min. 2. During the subsequent 13 min, 40% of the liver protein radioactivity disappeared. The rate of this process was already maximal 0.5 min after the injection of puromycin, with no apparent lag. 3. Evidence is presented that this phenomenon represents the selective degradation of puromycinyl-peptides: (a) the magnitude of this fraction corresponded to the calculated proportion of protein radioactivity in nascent chains at the time of the puromycin effect; (b) the size distribution of the proteins disappearing between 2 and 14 min was smaller than that of those retained at 14 min; and (c) when the injection of puromycin was delayed for 5 min, or when the leucine pulse was interrupted by the injection of cycloheximide (rather than puromycin), the fraction disappearing within 14 min was much smaller. 4. The degradation of puromycinyl-peptides was much slower in the rapidly growing livers of animals recovering from a protein depletion than in the protein-depleted controls. It is concluded that the large decrease in the overall rates of total liver protein degradation previously described during liver growth is a general phenomenon, also affecting the rate of scavenging of abnormal proteins.  相似文献   

11.
Rats were given intravenous injections of l-[1-14C]leucine. Twelve minutes after injection, testes, kidneys, livers, and hepatomas were excised rapidly from one group of animals bearing Morris hepatoma 5123tc. From a second group of rats, the blood was removed 90 min after injection. Tissue extracts and serum were divided into three portions each, and albumin was isolated from each of the three portions by one of three different methods.The three different isolation procedures were the following: (A) pretreatment of the tissue extracts and serum with bovine serum albumin and its specific antiserum and subsequent immunoprecipitation of the rat serum albumin, (B) direct immunoprecipitation followed by dissolving the precipitated rat serum albumin in acid/ethanol, precipitation with ether, and ion-exchange chromatography of the redissolved albumin on CM-cellulose, and (C) a modification of a procedure published previously including fractionation with trichloroacetic acid, ethanol, ether, and ammonium sulfate, chromatography on Sephadex G-100 and DEAE-cellulose, and preparative disc electrophoresis in polyacrylamide gel at pH 10.3 and pH 2.7.With method (A), radiochemically pure albumin can be obtained only from serum. Even though testis and kidney do not synthesize albumin, albumin preparations isolated by this procedure from these organs contain significant amounts of radioactivity. Specific radioactivities measured in albumin prepared by method (A) from the four tissues examined are 5–19 times larger than those in preparations isolated by method (C). Thus, method (A) is inappropriate for the isolation of radiochemically pure albumin from the tissues studied.Procedure (B) is sufficient to obtain radiochemically pure albumin from the serum as well as from the other tissues examined except liver. With liver, this method yields an albumin preparation containing 53% more radioactivity than does albumin isolated with method (C).Method (C) is the only procedure yielding radiochemically pure albumin from all sources, including liver. In liver and hepatoma, the properties of the radioactive impurities are very similar to the properties of albumin itself. Therefore, several purification steps and a careful analysis of the distribution of radioactivity among the albumin fractions after chromatographies and electrophoreses are necessary to separate radioactive impurities from the albumin from homogenates of these two sources.  相似文献   

12.
We have labelled the rat vitamin D binding protein (DBP), DBP-actin and rat albumin with 125I-tyramine-cellobiose (125I-TC). In contrast with traditional 125I-labelling techniques where degraded radioactive metabolites are released into plasma, the 125I-TC moiety is trapped intracellularly in the tissues, where the degradation of the labelled proteins takes place. By using this labelling method, the catabolism of proteins can be studied in vivo. In this study we have used this labelling technique to compare the tissue uptake and degradation of DBP, DBP-actin and albumin in the rat. DBP-actin was cleared from plasma at a considerably faster rate than DBP. After intravenous injection of labelled DBP-actin complex, 48% of the radioactive dose was recovered in the liver after 30 min, compared with 14% when labelled DBP was administered. Only small amounts of DBP-actin complex were recovered in the kidneys. In contrast with the results obtained with DBP-actin complex, liver and kidneys contributed about equally in the uptake and degradation of DBP determined 24 h after the injection. When labelled DBP was compared with labelled albumin, the amount of radioactivity taken up by the liver and kidneys by 24 h after the injection was 2 and 5 times higher respectively. In conclusion, liver and kidneys are the major organs for catabolism of DBP in the rat. Furthermore, binding of actin to DBP enhances the clearance of DBP from circulation as well as its uptake by the liver.  相似文献   

13.
1. The isolated microsome fraction of regenerating rat liver was incubated with cell sap, a source of energy and [35S]methionine, [14C]isoleucine or [14C]leucine for different periods of time, and microsomal albumin isolated. 2. The distribution of these isotopes in albumin was determined by separation of tryptic peptides from the protein. Radioactivity was measured in peptides either qualitatively by radioautography or quantitatively by labelling with both 3H and 14C. 3. A gradient of radioactivity existed at all times in albumin isolated after incubating microsomes. 4. The shorter the incubation time the fewer the peptides labelled in albumin, but the peptides with highest specific activity after short incubation times corresponded to those with highest specific activities after long incubation times. 5. Leucine released from the C-terminus of albumin had a higher specific activity than the mean specific activity of the remaining leucine residues in albumin. 6. The peptide with the highest specific activity in albumin is probably derived from the C-terminus of the protein. 7. [14C]Glutamic acid is incorporated into the N-terminus of albumin after incubating the microsome fraction with this isotopically labelled amino acid, cell sap and a source of energy. The specific activity of the N-terminal glutamic acid under these conditions is less than the mean specific activity of the remaining glutamic acid and glutamine residues in albumin. 8. The results are interpreted as reflecting a sequential synthesis of serum albumin in the isolated microsome fraction of rat liver. The direction of synthesis of albumin is from the N-terminus towards the C-terminus. 9. The bulk of incorporation of radioactive amino acid into albumin in the isolated microsome fraction is due to completion of partially completed, pre-existing peptide and polypeptide chains. A limited synthesis of new chains of albumin does, however, occur.  相似文献   

14.
The effects of an inflammatory insult on albumin of the rat liver were investigated at the cellular level and were correlated with serum albumin concentration. After SC injection of turpentine, the livers were perfused and fixed in vivo; serial liver sections were stained using a streptavidin-ABC-immunoperoxidase technique with an antibody to rat albumin. Albumin and total protein were measured at intervals after turpentine injection in whole livers and in serum. Fibrinogen was determined in plasma only. Twenty-four hours after turpentine injection serum albumin had dropped by 25% and was at 50% of its initial value at Day 3. Serum fibrinogen increased 2.4-fold within 24 hr and decreased thereafter. Liver homogenates showed no significant changes in albumin concentration. Immunohistochemically, all hepatocytes stained positive for albumin in normal animals. During inflammation, the immunostainable albumin content vanished entirely in a majority of all hepatocytes while remaining unchanged in other cells, thus producing a strikingly patchy staining pattern. No signs of resumption of albumin accumulation in depleted hepatocytes were seen after 8 days, despite a clear trend towards normalization of serum albumin concentration. These results suggest that individual hepatocytes differ widely in their response to agents that suppress albumin synthesis in an acute-phase reaction.  相似文献   

15.
In liver cells recovering from reversible ischaemia, total protein synthesis by postmitochondrial supernatant and membrane-bound and free polyribosomes is not different from that in sham-operated controls. However, the relative proportion of specific proteins is changed, since the incorporation of [3H]leucine in vivo into liver albumin, relative to incorporation into total protein, as determined by precipitation of labelled albumin with the specific antibody, decreases by 40-50% in post-ischaemic livers. Cell-free synthesis by membrane-bound polyribosomes and poly(A)-enriched RNA isolated from unfractionated liver homogenate shows that the decrease in albumin synthesis in liver of rats recovering from ischaemia is due to the relative decrease in translatable albumin mRNA.  相似文献   

16.
After a single injection of formaldehyde-treated 131 I-albumin into the heart, the incorporation of the labelled protein by liver (% of total injected radioactivity/% of body weight of the organ) was far greater than in other organs. In kidney and spleen it was respectively six and three times greater than in lungs, intestine, testis and fatty body. No radioactivity was found in brain. The radioactivity in liver and kidney reached a peak 30 minutes after the injection, and quickly decreased during the following four hours. In the 27,000 g × ten minute particles recovered from liver homogenates of animals sacrificed at various times after injection, the rate of 131 I-albumin hydrolysis in vitro and the percentage of trichloroacetic acid soluble radioactivity at zero time of incubation showed different stages of intraparticulate hydrolytic activity. The incorporation and intraparticulate hydrolysis in toad kidney was very low if compared with that of toad liver or mouse kidney; however the catheptic specific activity in toad kidney doubles that of mouse kidney. Isolated toad liver was perfused with total blood, containing 131 I-albumin, for five hours at 22°C in a special chamber. In this conditions, 16% of the labelled albumin was hydrolyzed by the liver.  相似文献   

17.
Albumin was isolated immunologically from various subcellular fractions from livers of adult male rats receiving an intraperitoneal injection of [3H]leucine to investigate the kinetics and pathway of subcellular transfer of newly synthesized albumin during secretion. At appropriate time intervals, livers were excised and fractionated into endoplasmic reticulum and Golgi apparatus. Golgi apparatus were further subfractionated into cisternae and secretory vesicles. In endoplasmic reticulum fractions, labeled albumin appeared within 7.5 min of injection of isotope, followed by a rapid decline in specific activity. Albumin in Golgi apparatus was labeled and concentrated in secretory vesicles over 25 min. The radioactivity in albumin per mg total protein was highest in secretory vesicles and insignificant in the cisternal fraction. Labeled albumin was present in serum by 30 min and radioactivity in serum albumin reached a plateau within 60–90 min after injection of isotope. Results provide evidence for the migration of albumin from its site of synthesis on endoplasmic reticulum membrane-bound polyribosomes to its site of secretion into the circulation via the Golgi apparatus. The pathway of albumin transport to secretory vesicles is suggested to involve peripheral elemenst of the Golgi apparatus. Secretory vesicle formation and maturation required 20 to 30 min for completion, via a mechanism whereby the inner spaces of the central saccules may be bypassed.  相似文献   

18.
The axonal transport of labelled proteins was studied in the optic system of adult rabbits after an intraocular injection of [3H]Ieucine. It was demonstrated that the precursor was incorporated into protein, which was transported along the axons of the retinal ganglion cells. Intraocularly injected puromycin inhibited protein synthesis in the retina and markedly inhibited the appearance of labelled protein in the optic nerve and tract. It was further demonstrated by intracisternal injection of [3H]leucine that an intraocular injection of puromycin did not affect the local protein synthesis in the optic nerve and tract. Cell fractionation studies of the optic nerve and tract showed that the rapidly migrating component, previously described as moving at an average rate of 110-150 mm/day, was largely associated with the microsomal fraction. About 40 per cent of the total protein-bound radioactivity in this component was found in the microsomal fraction and about 15 per cent was recovered in the soluble protein fraction. Most of the labelled material moving at a rate of 1-5-2 mm/day was soluble protein. The specific radioactivity of this component was about ten times greater than that of the fast one. In the slow component about 50 per cent of the radioactivity was found in the soluble protein fraction and about 10 per cent of the radioactivity was recovered in the microsomal fraction. Radioautography demonstrated incorporated label in the neuropil structures in the lateral geniculate body as early as 4-8 hr after intraocular injection. The labelling of the neuropil increased markedly during the first week, and could be observed after 3 weeks.  相似文献   

19.
以氯胺T为氧化剂参照Hunter和Greenwood的多肽类化合物的碘化标记法,制备了~(125)I—标记的蜂毒肽在小鼠体内分布、吸收、排泄的研究,实验证明小鼠肌注~(125)I—蜂毒肽后,吸收很快,主要分布部位为肾、肺、心、肝、小肠、关节、脾与肌肉,脑组织中含量很少,肌肉注射后5分钟血液中含量可达70%,~(125)—I蜂毒肽主要经肾排泄,肌注后30分钟肾脏浓集最高,而尿液中以1.5小时为最高,而粪便中排泄少。  相似文献   

20.
1. Human kidney alpha-D-mannosidase (form A) was labelled with 125I to a specific radio-activity of approx. 2250muCi/mg of protein, essentially without loss of enzymic activity. The enzymic activity and radioactivity of the iodinated material also co-migrated in gel filtration and gel electrophoresis. 2. The binding of 125I-labelled mannosidase in vitro to particulate material in liver and kidney homogenates was of the other of 2 pg/mg of particulate material in liver and kidney homogenates was of the order of 2pg/mg of particulate protein withing 16h at 37 degrees C, and essentially zero in intervals of up to 60 min. The degradation in vitro of labelled exogenous mannosidase was of the order of 10-20pg/ 16th per mg of protein in postnuclear supernatant, and it was saturated entirely within 1h at 37 degrees C. 3. The binding of labelled mannosidase in vivo to particulate elements of liver homogenates 60 min after intravenous injection was at least 10 times higher in terms of specific radioactivity than the highest value attainable in vitro. Virtually all exogenous enzyme bound to liver particulate material could be recovered in macromolecular form after disruption of membranes by detergents. 4. The radioactive enzyme bound to liver particulate material could be detached almost completely by shearing, repeated freezing and thawing, and exposure to strong detergents under conditions that do not eliminate rough-endoplasmic-membrane structure. It could bot be released, however, by high salt concentration (0.5M-KC1) or by exposure to weak detergents such as Tween 80. The particle-bound enzyme should thus be associated with plasma membranes and lysosome-like elements. 5. Of the rat tissues studied, only liver could approach, within 60 min after the injection, the concentration of exogenous mannosidase found in the blood serum. The activity per g tissue weight fell progressively from liver (60% of serum value) to kidney (16% of serum value), lung (8% of serum vlaue), spleen (6% of serum value) and brain (0.9% of serum value). Most of the radioactive enzyme found in tissues other than liver appeared to be present in a free form, whereas in liver more than 50% of the labelled enzyme was associated with membrane elements.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号